The goal of this research is to exploit techniques for the targeted manipulation of neural activity to identify, and functionally define, brain networks underlying specific behaviors. As a model for such investigations, we are identifying the networks that govern the behavioral program executed by adult fruit flies shortly after emergence from the pupal case. We have demonstrated that this program consists of two principle phases, an adaptive behavioral phase, which mediates the search for a suitable environment, and an environmentally-insensitive phase, which drives expansion of the wings to make them flight-worthy. Focusing on the innate, environmentally-insensitive phase, we have also shown that two anatomically and functionally distinct groups of neurons contribute to wing expansion. Work that we are preparing for publication further elucidates the functional roles of these groups and the key role played by the hormone bursicon. In addition, we have initiated experiments to identify the sensory pathways responsible for the adaptive, environmentally-sensitive phase of behavior. Elucidation of the circuits underlying both phases of the post-emergence behavioral program promises a detailed understanding of how intrinsic and extrinsic factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Identification of the mechanisms by which neuronal networks interact and adapt to organize behavior should shed light on the deficits in behavioral organization that lie at the root of many mental disorders, including obsessive-compulsive disorder, schizophrenia, and bipolar disorder. Specific details of our accomplishments over the last year follow. Our primary efforts over the past year have focused on the mechanisms that govern the environmentally-insensitive phase of the post-emergence behavioral sequence of Drosophila. Capitalizing on our previous identification of a command system for wing expansion (Peabody et al., 2009), we have been addressing three principal questions: 1) At what developmental stage does the command system become competent to drive wing expansion;2) What is the minimal cell group within the command system that is sufficient to activate the wing expansion program;and 3) What neuronal effectors lie downstream of the command system. We have made considerable progress on each of these questions and are currently preparing two manuscripts describing our results. Consistent with our earlier suppression studies, we have identified the 16 neurons that express the hormone bursicon as essential components of the command system: Targeted stimulation of these neurons by UAS-TRPM8 elicits the entire wing expansion motor program in newly eclosed flies. Interestingly, flies that have not yet eclosed do not respond to stimulation either by activation of the wing expansion program or by releasing bursicon. This observation suggests that the bursicon-expressing neurons are inhibited prior to eclosion and that eclosion, or some process closely correlated with it, gates the activity of the command system. Remarkably, we also find that the wing expansion program can be driven by stimulation of a single pair of the bursicon-expressing neurons located in the subesophageal ganglion (i.e. the BSEG), indicating that these two neurons act as command neurons for wing expansion. The identification of the BSEG as command neurons for wing expansion was made possible by combining our UAS-TRPM8 technique for neuronal activation, which we have also used to study motivation and memory retrieval systems in collaboration with the Waddell laboratory (Krashes et al., 2009), with our Split Gal4 system for refined transgene targeting (Luan et al., 2006). The Split Gal4 system is a combinatorial method that relies on the independent targeting of the two component domains of the Gal4 transcription factor: the DNA-binding (DBD) and transcription activation (AD) domains. Each domain is fused to one of two complementary, heterodimerizing leucine zippers so that the DBD and AD domains associate in cells that express both. In these cells, and in these cells alone, transgenes downstream of Gal4s UAS binding site are expressed. We previously demonstrated that this system could be used in flies to label neurons at the intersection of two overlapping expression patterns (Luan et al., 2006). We are now collaborating with the Allen Institute for Brain Science to determine whether the Split Gal4 technique might be similarly used in transgenic mice. We have demonstrated in preliminary, proof-of-concept studies that we can reconstitute Gal4 transcriptional activity in transgenic mice using the Split Gal4 system. We are currently evaluating the ability of the system to drive restricted, intersectional UAS-transgene expression. To identify effectors of the command system for wing expansion, we have focused on identifying neurons that express the bursicon receptor, which is encoded by the rickets gene. In order to concomitantly characterize the functions of rickets-expressing neurons, we have used homologous recombination to insert the Gal4 gene into the putative translation start site of the rickets gene. Such "rickets-Gal4" driver lines can, in principle, be used to identify rickets-expressing neurons by driving the expression of reporter transgenes, such as UAS- EGFP, and to functionally manipulate them by driving expression of effector transgenes, such as UAS-TRPM8. We have thus far generated two Gal4 knock-in lines, using constructs with and without artificial transcription termination signals and are currently in the process of characterizing their respective efficacies. In general, investigation of the circuits that govern posteclosion behavior in Drosophila using the palette of tools we have developed should provide insight into the principles used by all nervous systems to generate and organize behavior. In addition, our work should serve as a proof of concept of a circuit mapping approach that can be extended to studies of mammalian behavior as similar tools become available for vertebrate organisms. Indeed, one of our goals is to extend those technologies that we find useful in the fly to mammalian model systems.